Abstract Hematopoiesis must be sustained throughout life in order to maintain immune response capabilities and to replenish blood supplies. A large proportion of adult HSCs therefore remain quiescent, rarely undergoing cell division, until they become activated upon bodily insults, such as infection and blood loss. These quiescent HSCs must undergo global changes in gene expression patterns in order to leave their dormant state and differentiate. Chromatin modifiers that act independently of cell division may therefore be of particular importance in regulating the behavior of quiescent HSC through the establishment of the chromatin landscape differentially across the genome. One such chromatin modifier is the histone variant H3.3 chaperone Hira, which is responsible for incorporating H3.3 independent of DNA replication at poised and transcriptionally active promoters. Despite the fact that this histone variant only differs from the conventional H3 histones by four or five amino acids, its gene-specific association is required for proper establishment of gene expression and lineage specification in murine embryonic stem cells (mESC), post-mitotic neurons, and oocytes. Since Hira deposits H3.3 independently of S phase, we are testing the hypothesis that Hira plays a critical role in the maintenance of quiescent HSCs, since quiescent HSCs will need cell division-independent mechanisms to regulate H3.3. The early results from this study demonstrate a requirement for the histone chaperone Hira in HSC quiescence and self-renewal behaviors. We will next assess the ability of Hira-mediated H3.3 incorporation to maintain normal HSC activity and gene expression patterns. Knowledge gained from this study regarding in vivo HSC epigenetic state regulation may inform future research for clinical applications, such as for improved methods of in vitro HSC expansion. Characterizing the importance of H3.3 incorporation and turnover by Hira in normal HSC activity will therefore provide invaluable information regarding the epigenetic regulation of hematopoietic homeostasis. Rebecca Murdaugh will conduct the research proposed in this application over the course of 4 years. She is a graduate student in the second year of her PhD with undergraduate training in biochemistry and cell and molecular biology.